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Feeding frequency and water temperature impact apparent digestibility coefficients of sablefish (Anoplopoma… Pace, Steven Alan 2013

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FEEDING FREQUENCY AND WATER TEMPERATURE IMPACT APPARENT DIGESTIBILITY COEFFICIENTS OF SABLEFISH (Anoplopoma fimbria) by STEVEN ALAN PACE B.Sc., Queen’s University, 2009 Fisheries and Aquaculture Diploma, Vancouver Island University, 2010  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in The Faculty of Graduate Studies (Animal Sciences)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) February, 2013  © Steven Alan Pace, 2013  ABSTRACT Integrated multi-trophic aquaculture (IMTA) is a sustainable form of polyculture that holds promise in alleviating sustainability and environmental concerns facing current aquaculture practices. For sablefish (Anoplopoma fimbria) to be considered for use in IMTA systems, information on the digestibility of the diets by this species is needed. These data are critically important in establishing the necessary relationships between various species in IMTA networks. The effects of feeding frequency and water temperature on the apparent digestibility coefficients of juvenile sablefish were assessed in two separate experiments. In experiment 1 the effect of feeding frequency on apparent digestibility of a commercial feed (supplemented with chromic oxide as an indigestible marker) was determined by feeding juvenile sablefish either once, twice, or three times per day. In experiment 2 the effect of water temperature on apparent digestibility coefficients of the feed was determined in groups of sablefish reared at 8 ºC and 11.5 ºC. In both experiments the feed and faeces were analyzed for chromic oxide, ash, moisture, protein, and energy content and the resulting data were used to calculate apparent digestibility coefficients (ADC) for crude protein (CP), gross energy (GE), and organic matter (OM). Results from Experiment 1 indicate that feeding frequency had an effect on all ADC; with lower values of digestibility found with higher feeding frequencies (p ≤ 0.05). In Experiment 2 fish maintained at 11.5 ᵒC had higher ADC compared to those at 8.0 ˚C (p ≤ 0.05). This new information will be applied to a future IMTA ecosystem model, in an effort to enhance system efficiency.  ii  PREFACE  Chapter 3 is based on work conducted at the Centre for Aquaculture and Environmental Research (CAER) laboratory, located at 4160 Marine Drive, West Vancouver, BC. I was responsible for caring of the sablefish used in the experiments, and completed “The Experimental Fish Online Training Program” from the Canadian Aquaculture Institute (Centre for Life-Long Learning at the University of Prince Edward Island). The experiments conducted fell under the AUP of UBC-CCAC, certificate #A10-0051. I was also responsible for modifying all the tanks where the sablefish were held for the duration of the project. Dr. R. Scott McKinley, Dr. Shannon Balfry, Dr. Ian Forster, and Dr. Nina von Keyserlingk helped me with the design and implementation of my exposures. William Wong, Jonathan Grass, Bianca Arney, Jenna Radloff, Lorena Garcia, and Mahmoud Rowshandeli aided me when sampling, measuring, or euthanizing fish was necessary. Dr. Ian Forster and Mahmoud Rowshandeli aided me with all the analyses which took place. Dr. Shannon Balfry and Dr. Ian Forster helped me with any statistics involved with this project.  iii  TABLE OF CONTENTS  Abstract………………………………………………………………………………..………  ii  Preface………………………………………………………………………………………… iii Table of Contents……………………………………………………………………………..  iv  List of Tables………………………………………………………………………………….  vi  List of Figures………………………………………………………………………………… vii Acknowledgements…………………………………………………………………………… viii Dedication……………………………………………………………………………………..  ix  Chapter 1: Introduction ....……………….…………………………………………………..  1  Chapter 2: Measuring digestibility…………….…………………………………………….  6  2.1 Introduction……………………………………………………………………….. 2.2 Faecal collection techniques……………………………………………………… 2.2.1 Dissection……………………………………………………………….. 2.2.2 Manual stripping………………………………………………………... 2.2.3 Collection from water column…………………………………………... 2.3 Comparison of faecal collection techniques………………………………………. 2.3.1 Direct method comparisons……………………………………………... 2.3.2 Indirect method comparisons……………………………………………. 2.4 Conclusion …………………………………………………………………………  6 8 8 9 10 13 13 15 16  Chapter 3: Feeding frequency and water temperature impact apparent digestibility coefficients of sablefish (Anoplopoma fimbria)………………………………... 18 3.1 Introduction………………………………………………………………………... 3.2 Materials and methods…………………………………………………………….. 3.2.01 Experimental fish……………………………………………………….. 3.2.02 Faecal collection……………………………………………………….. 3.2.03 Feed manufacturing…………………………………………………….. 3.2.04 Feeding frequency trial…………………………………………………. 3.2.05 Water temperature trial………………………………………………… 3.2.06 Analyses………………………………………………………………… 3.2.07 Moisture, ash, chromic oxide…………………………………………… 3.2.08 Protein…………………………………………………………………... 3.2.09 Energy…………………………………………………………………...  18 20 20 21 22 22 23 23 23 24 25 iv  3.2.10 Organic Matter…………………………………………………………. 3.2.11 Apparent digestibility coefficient (ADC) calculations………………….. 3.2.12 Statistics………………………………………………………………… 3.3 Results……………………………………………………………………………… 3.4 Discussion…………………………………………………………………………..  25 25 26 26 27  Chapter 4: Figures and Tables ................................................................................................ 33 Chapter 5: General Conclusion ..……………………………………………………………. 38 References ...…………………………………………………………………………………… 41  v  LIST OF TABLES Table 1. The effect of feeding frequency and rearing temperature on the apparent digestibility coefficients of juvenile sablefish fed a commercial diet……………………………………….. 37  vi  LIST OF FIGURES Figure 1. The locations where pressure is applied to the fish when conducting manual stripping, as described by Austreng (1978)…………………………………………………………………... 33 Figure 2. The system developed by Choubert et al. (1982). Effluent water, containing faeces (9), flows over a series of moving screens (1). As the unit progresses, faeces dry and are then deposited into a collecting tray (11)……………………………………………… 34 Figure 3. The system developed by Hajen et al. (1993). Effluent water flows into the adjacent settling column and faeces collect at the base of the unit……………………………………… 35 Figure 4. Schematic of faecal collection system when attached to tank………………………………….. 36  vii  ACKNOWLEGEMENTS This thesis would have been impossible without the help of many amazing individuals. I would like to thank my supervising committee of Dr. R.S. McKinley, Dr. S. Balfry, Dr. I. Forster, and Dr. N. von Keyserlingk for all of their support and guidance over the past two years. I would also like to thank NSERC for funding my work as well, and CIMTAN, in particular Dr. G. Reid, for providing me with such a wonderful research opportunity. Countless other individuals helped me during the course of my project and I would like to quickly thank as many of them as possible. To William Wong and Mahmoud Rowshandeli; thank you for helping me feed my fish, count, sort, perform all the analyses, and in general make my life manageable. To Lorena Garcia, Jenna Radloff, Kate Cummins, Bianca Arney, and Jonathan Gross; I’d like to thank you for helping me whenever I asked for people to feed fish for me and help me with sampling days. Thank you all so much! Finally I would like to thank my family for their endless support and helping me revise this thesis as well. Thank you all so much!  viii  DEDICATION  To all my friends and family  ix  CHAPTER 1: INTRODUCTION The global human population is expected to exceed 9 billion by 2050, adding in excess of 2 billion people to the planet in less than 40 years (Heilig et al., 2012). This dramatic increase in population could pose monumental challenges for the human race, especially concerning food and water consumption. Competition for land, water, and energy combined with the overexploitation and mismanagement of many fisheries hinders the ability to produce enough food to feed a growing population (Godfray et al., 2010). To meet rising demand, global food production would need to increase by 70% by 2050 (World Bank, 2008; Royal Society of London, 2009). Reaching this estimate may be quite difficult, especially considering that agricultural land in industrial countries has decreased by 3% from 1961-2007 (Royal Society of London, 2009). Furthermore, agricultural productivity has decreased in recent years (Royal Society of London, 2009; von Braun, 2007). With the problems humanity may experience in the future, how will we be able to feed such an increase in population? A potential solution which can assist global food shortages in the future may be aquaculture (Godfray et al., 2010). There are many different types of aquaculture including open-water, closed containment systems, re-circulating aquaculture systems, and polyculture. The industry was worth US$108 billion in 2008, with 80% of the revenues arising from Asian markets (Bostock et al., 2010). Aquaculture is the fastest growing food sector in the world (Bostock et al., 2010), with annual growth rates of 6.6%. The development of the aquaculture sector may alleviate pressure on exploited wild fisheries, where 57% of world-wide fisheries are fully exploited and an additional 29% are over-exploited (FAO, 2012). However sector growth has been hindered due to increasing environmental and political pressures. The main criticisms  1  of aquaculture are related to environmental sustainability, disease control, and waste management (Ayer and Tyedmers, 2009). A number of novel approaches to develop sustainable open-water aquaculture methods have been suggested in recent years. These include closed containment operations, submerged sea cages, and polyculture. Closed containment aquaculture operations attempt to eliminate any risks associated with the contact of cultured organisms with wild species by establishing a barrier between the two (Fredriksson et al., 2008). Closed containment operations include marine floating bag and cement systems, and land-based re-circulating aquaculture systems (RAS) (Ayer and Tyedmers, 2009) and are effective at reducing many local ecological impacts associated with open net pen marine farms, including waste production, escapees, and disease control (Neori et al., 2004). However the amount of resources and energy required to operate a closed containment system may hinder any widespread adoption by the industry (Ayer and Tyedmers, 2009). Traditional polyculture practices combine agriculture and aquaculture for use within the same system, with certain crops utilizing the waste from other crops in the system (Neori et al., 2004). Polyculture is an excellent means to improve sustainability, however overall yield is typically sacrificed for a more sustainable operation (Neori et al., 2004). An alternative method of polyculture is known as Integrated Multi-Trophic Aquaculture (IMTA). This approach may help alleviate many environmental and sustainability concerns associated with traditional open net pen aquaculture (Soto, 2009). IMTA is the practice of coculture of species inhabiting different trophic groups within the same system (Soto, 2009). The nutrients in the by-products or wastes from one species can be used as inputs for other species of lower trophic levels (Reid et al., 2011). Fed species of top trophic level are finfish (Oncorhynchus, Salmo, Anoplopoma), which are cultured with extractive aquaculture species  2  (suspension feeders, detritovores: Mytilus, Halotis, Crassostrea, Cucumaria) and inorganic extractive species (macroalgae: Laminaria, Macrocystis) (Soto, 2009). These species are present in the system at a specific ratio which, considered in combination with site location, species selection, and other factors (such as regulations) reduces the environmental impact of the operation (Reid et al., 2011; Soto, 2009). Ideally IMTA farms balance waste production with nutrient extraction, allowing operations to reduce their environmental impact and remain economically viable (Neori et al., 2004). Furthermore, since only the finfish are fed in IMTA the other extractive species in the operation are sources of extra profit (once they are stocked). Nutrient flow between trophic levels is very important in IMTA. Finfish are first stocked into their enclosures and fed a diet. Undigested particles or faeces can be taken up by the organic extractive species present in the system (Mytilus, Halotis, Crassostrea). Other nutrients produced by the finfish such as ammonia and phosphates can be absorbed by the inorganic extractive species (Laminaria, Macrocystis). Furthermore, any uneaten feed pellets or wastes which fall below the finfish enclosures can be consumed by detritovores (Cucumaria). In IMTA, the organic and inorganic extractive species are supplementing their traditional diet with faeces and nutrients produced from the sablefish. Therefore the wastes associated with finfish net pens in IMTA systems can be reduced. Finally, the species used in an IMTA farm are typically aligned with the major water and current flow going through the farm in order to maximize the amount of wastes and nutrients which can be taken up by the organic and inorganic extractive species. One particular species of finfish which is being investigated for use in IMTA is sablefish (Anoplopoma fimbria). Sablefish can also be referred to as black cod, blue cod, bluefish, candlefish, coal cod, and coalfish. In spite of “cod” being included in many of the common names associated with the species, sablefish are members of the Scorpaeniformes order of fishes  3  and Anoplopomatidae family. Sablefish are deep sea fish that are distributed from the north Pacific along the Bearing Sea coasts of Kamchatka, Russia, and Alaska southward to Hatsu Shima Island, southern Japan, central Baja California, and the northern coasts of Mexico. Sablefish can be found at depths of 0-2740 meters, live up to 65 years of age, and grow to lengths of 120 centimeters. Juveniles are typically pelagic and found on the surface and near shore waters, while adults are demersal and reside on mud bottoms anywhere from 305-2740 m in depth. Sablefish consume crustaceans, worms, and small fishes (Hart, 1973). Sablefish are coveted, particularly in Japan, for their soft, oily, and white flesh. In order for IMTA to be successful, the digestibility of diets fed to sablefish must be known. The Canadian Integrated Multi-Trophic Aquaculture Network (CIMTAN) is assimilating knowledge from multiple-disciplines and fields pertaining to IMTA to develop an ecosystem model which can be used for constructing IMTA systems (Chopin, 2011). In order to optimize the design of IMTA sites, CIMTAN is collecting quantitative, qualitative, spatial, and temporal data on the nutrients loaded from upper tropic levels of the systems (Soto, 2009). To fully comprehend how efficient animals are at digesting their food and what nutrients are present in fish faeces, diet digestibility trials must be conducted. Digestibility experiments are critical for the manufacture of optimal and efficient feeds, along with aiding IMTA ecosystem modeling. In order to determine the diet digestibility of sablefish feed, faeces must be collected in order to calculate apparent digestibility coefficients (ADC). ADCs can illustrate how efficiently the fish is digesting certain nutrients from the feed. Therefore, performing diet digestibility trials is an excellent means to collect quantitative data on the nutrients loaded from sablefish. The objective of this research project was to determine the impact of feeding frequency and water temperature on sablefish apparent diet digestibility. Fish feed is the greatest expense  4  for aquaculture farms, and feeding frequency has been shown to play an important role in improving feed efficiencies. Furthermore, feeding frequency can directly impact feed digestibility. Variations in rearing temperatures can also affect diet digestibility and feeding efficiencies (Schurmann and Steffensen, 1997). It was therefore important to understand how feeding frequency and water temperature influence diet digestibility and hence nutrient quality of sablefish faeces, to assess potential species interaction in an IMTA system.  5  CHAPTER 2: MEASURING DIGESTIBILITY  2.1 Introduction To fully comprehend how efficient animals are at absorbing their food, one must understand diet digestibility. Digestibility is important to understand for the manufacture of optimal animal feeds, estimating energy gains in animals, determining potential trophic relationships between species, and critical to the economical use of animal feeds, which can be half of production costs. Furthermore, diet digestibility information is required for co-culture operations, such as integrated multi-trophic aquaculture (IMTA), because these systems rely on nutrient flow from the organisms cultured within the system. It is prudent to investigate diet digestibility values for all cultured species in order to be able to develop efficient, yet inexpensive feeds and fully comprehend the impact one species may have on others in a coculture system. There are a variety of indirect and direct methods for investigating diet digestibility (Maynard and Loosli, 1969). Indirect diet digestibility trials typically use an indigestible marker (such as chromic oxide, Cr2O3) that is combined with the animal feed. After faeces have been expelled from the animal, the ratio of the marker remaining in the faeces compared to what was originally in the feed is used to calculate the apparent digestibility. In contrast, direct diet digestibility trials are much more difficult to conduct, mainly because the exact amount of feed used and faeces produced by the animal must be quantified. Since faeces can easily leach and mix with feed in aquatic systems, direct assessment studies must be well organized and carefully conducted.  6  Diet digestibility can also be estimated by conducting feed trials or by isolating specific enzymes in the gastro-intestinal tract. These two techniques are classified as in vitro and in vivo methods. In vitro apparent digestibility studies typically isolate one (or more) enzymes from the digestive tract of an animal that are combined with protein/feed slurries in test tubes (Grabner, 1985). This approach allows many in vitro investigations to be performed in a relatively short period. In contrast, in vivo investigations involve performing feed trials on live animals, which are either sacrificed at the end of the feed study, or their faeces are collected during the feed study (Cho and Slinger, 1979; Hajen et al., 1993). Typically in vivo studies take much longer to perform than in vitro studies because feed trials can take weeks or months to complete. Gomes et al. (1998) suggested that if faeces can be collected efficiently, in vivo trials may be better suited for obtaining apparent digestibility values for fish because in vivo investigations better mimic real life conditions of fish. Faecal collection trials conducted with terrestrial animals are relatively easy to perform because faeces are deposited on land, are very easy to collect, and are subject to minimal leaching (Austreng, 1978). In aquatic species, it is much more difficult to collect and quantify faeces due to the amount of leeching faeces experience once they enter the water (Windell et al., 1978; Satoh et al., 1992; Hajen et al., 1993). Additionally, faeces deposited in water can rapidly breakdown which can complicate faecal collection (Austreng, 1978), or be mistaken for uneaten feed pellets. Due to these factors, collecting faeces in aquatic systems is challenging, and therefore a variety of techniques have been designed to adequately collect faeces.  7  2.2 Faecal collection techniques Faeces must be collected if one wishes to quantify various diet digestibility components (such as protein, lipids, carbohydrates, ash, etc.) or calculate apparent digestibility coefficients (ADC). There are three principal methods of collecting faeces in aquatic systems; dissection, stripping, and collection of excreted faeces from the water column (Glencross, 2007). A variety of other techniques have been attempted, such as anal suction (Windell et al., 1978) and placing fish in metabolic chambers (Post et al., 1965; Smith, 1971) but have received considerable criticism due to invasiveness associated with the techniques (Vandenberg and De La Noue, 2001). A much less invasive technique is the use of an inert marker, such as chromic oxide (Cr2O3), which is combined with the feed at a ratio of 0.5% to allow digestibility coefficients to be calculated.  2.2.1 Dissection Collection of digesta (not considered faeces because faeces must be expelled from the body) via dissection is typically conducted following other faecal collection techniques (stripping, suction, collection from water column, etc.) and involves euthanizing the fish. Digestibility for protein, fats, carbohydrates, ash, and gross energy increases as digesta moves through the gastro-intestinal tract (Austreng, 1978). Obtaining digesta via the dissection method is an excellent way of obtaining relatively pure and unaltered fish digesta, prevents leaching of nutrients into the water column, and avoids contamination from fish pellets (Austreng, 1978). Furthermore, fish tanks do not need to be modified in any way when collecting digesta, which could be quite important given the space or funds available for a given digestibility project.  8  The major disadvantage of the dissection method is that fish must be sacrificed to obtain any digesta. Therefore dissections typically take place at the conclusion of most digestibility studies. When combined with other collection methods, dissection can help strengthen results and reveal any potential errors with other collection methods used.  2.2.2 Manual stripping Stripping fish for their faecal contents was first performed by Nose in 1960, but since then this method has been widely adopted (e.g., Singh and Nose, 1967; Nose, 1967; Austreng, 1978). When sampling fish faeces by stripping, a small amount of pressure is applied to the fish halfway between the pectoral and ventral fins, and is continued towards the anus (Austreng, 1978) or alternatively pressure is only applied from the ventral fins to the anus (Singh and Nose, 1967; Nose, 1967) (Figure 1). According to Austreng (1978), when paired with an inert marker like chromic oxide, manual stripping is the most accurate and convenient technique for collecting fish faeces. Manual stripping provides many advantages over other digestibility methods, namely fish do not need to be sacrificed, and can also live in normal culture conditions for the duration of the trial. In addition feed consumption and faecal output do not necessarily need to be quantified (Austreng, 1978). However, manual stripping of digested contents is not possible on some species of fish, juvenile fish, and crustaceans (Glencross, 2007). Manual stripping is an easy and inexpensive means to collect fish faeces. It can be performed on a wide range of fish, and can be used in most apparent digestibility experiments. Another advantage to this method is that fish tanks do not need to be modified for faeces collection. The major criticism of manual stripping is related to the distress, and possible death,  9  due to the handling. Although anesthesia has been reported to calm fish, it can cause fish to immediately expel faeces (Spyridakis et al., 1989).  2.2.3 Collection from water column Before the early 1970’s, collecting faeces from aquatic organisms was either conducted manually by stripping faeces from live or dead fish (Nose, 1960), or by placing fish in individual metabolic chambers (Smith, 1971). However, both these methods presented numerous difficulties for faeces collection, such as contamination and leaching of the faeces. Furthermore both techniques were quite stressful to fish as they require extensive handling and can therefore potentially confound results (Austreng, 1978). It became clear that an alternative, less stressful method for collecting fish faeces was needed. Cho and Slinger (1979) developed a novel method to collect fish faeces which would form the foundation for faeces collection in aquatic systems for many years. Normally referred to as the Guelph System, Cho and Slinger constructed units consisting of three tanks that all drained into a common effluent drain channel. This channel led to a separate cylinder known as the faecal collecting column where faeces would swirl in the water column and gradually settle at the base. A small valve located at the base of the faecal decanting column would allow the faeces to be easily removed from the system. Typically fish faeces would become trapped in the settling column within 2 minutes of being expelled from the fish (Cho et al., 1982). The Guelph collection system (Cho and Slinger, 1979) has many advantages over other methods. For example, fish do not need to be sacrificed (dissection) or excessively handled (manual stripping) in order to collect faeces. Furthermore faeces decant and collect in the base of  10  the system quite rapidly, thus reducing leaching. The system does not take up much space, making it a practical option for confined locations. However, since multiple tanks drain into a common decanting column, analyzing results at the per-tank level is impossible. The stand-pipe also reduces the effective settling area for faeces in the decanting column. Fish must be fed slowly and carefully in order to ensure that fish pellets do not mix with the faeces in the system. Finally, special tanks, with sloping bottoms and a long, narrow drain are required when constructing this system which may increase costs associated with any experiment. Another effective, yet vastly different, system for capturing fish faeces was developed by Choubert and colleagues (1982). Instead of relying on tank/plumbing design and modifications (Cho and Slinger, 1979; Hajen et al., 1993), Choubert and his colleagues built a separate system that would collect faeces from the effluent drain (Figure 2), using a conveyor belt to collect and dry fish faeces. Screens attached to the conveyor belt captured faeces as the effluent drain water passed over the screen so fish faeces would be removed from the water and begin to dry. When the screen reached the left side of the conveyor belt, it would flip over and all dry faecal matter collected on the screen would be deposited into a pan, which could then be easily analyzed. This system boasts a very high faecal collection rate of 99%. Also, since faeces may begin to leach in the water column within 10 minutes (Windell et al., 1978; De La Noue et al., 1980) the relatively short time faeces spend in the water before collecting on the screen is beneficial. Moreover, the potential for faeces to be crushed by drainage water in the decanting column developed by Cho and Slinger (1979) is avoided because effluent water falls from a low height onto the moving screens. However the complexity and bulkiness of this system hinder its widespread application in other systems, especially those where space is at a premium. Additionally, a great deal of  11  mechanical knowledge is required in order to properly build and calibrate the conveyor belt (Choubert et al., 1982). A different method to collect faeces by passing the effluent water from fish tanks through a filtration column (TUF) was described by Ogino and colleagues (1973) and later refined by Satoh et al. (1992). Known as the TUF column system, a 5x30cm stoppered tube is attached to slightly off-centre tanks via a 2.5cm siphon tube. Faeces are siphoned out of the base of the tank into the collection column, where they can gather and settle at the base of the column. Effluent water drains out of the top of the column via a smaller exit tube. When the collection column is properly set-up and water is siphoned consistently from the tank, the incoming flow rate of water into the tank determines the rate of water movement through the TUF column. Aside from collecting faeces by dip netting, siphoning faeces into an adjacent tube is one of the easiest and simplest methods to collect fish faeces from the water column. The system is quite easy to set up and can be adapted to almost any (small) tank. The equipment used is very common and inexpensive, which makes the TUF method quite attractive. However, the TUF system is difficult to use with larger fish in big tanks as the angle that tanks need to be tilted and the position of the siphon to prevent faeces from sliding down the bottom of the tank or be successfully siphoned into the test tube. Furthermore due to the proximity of the effluent water valve to the intake in the collection test tube, it is possible that some faeces may escape the collection system. Drawing upon the success of the initial Guelph System for collecting fish faeces (Cho and Slinger, 1979) Hajen and colleagues (1993) attempted to modify and improve the longstanding design and determine if such a design would function with salt-water reared fish. Instead of grouping multiple tanks into one collecting system, Hajen et al. modified each  12  individual holding tank for faeces collection (Figure 3). The Guelph System (Cho and Slinger, 1979) and the system developed by Hajen et al. (1993) share many similarities. Both systems have tanks with sloping bottoms, have adjacent faecal collecting cylinders, and have similar faecal collecting outlets. The major differences between the traditional Guelph System (Cho and Slinger, 1979) and the newer system by Hajen (1993) is that the latter system does not have an internal standpipe in the faecal settling column and each tank has its own faecal collection cylinder. Because each tank has its own settling column it allows for faeces to be better quantified per tank (and per fish), however it may take longer to collect an acceptable amount of faeces to analyze since the system is based around one tank. Like all water-based faecal collection systems, nutrient loss via leaching is the major concern with this system since faeces are deposited into the water and then settle in the base of the decanting column (Glencross et al., 2005).  2.3 Comparison of faecal collection techniques on ADC data collection There are a variety of concerns regarding each faecal collection technique, many which may have severe implications on final estimated diet digestibility values. These criticisms range from the quality of faeces obtained via manual stripping, to the effect of nutrient leaching into the water column for the collection techniques.  2.3.1 Direct method comparisons Vandenberg and De La Noue (2001) compared the faecal collection techniques used by Austreng (1978), Cho and Slinger (1979), and Choubert et al. (1982) on the apparent digestibility of a diet. Manual stripping (Austreng, 1978) always yielded the lowest and the decanting  13  column method (Cho and Slinger, 1979) gave the highest ADC values (Vandenberg and De La Noue, 2001). The conveyor belt method (Choubert et al., 1982) typically gave the intermediate ADC value. This trend typically occurred because of the amount of leaching seen from the decanting column method allows for higher ADC’s to be estimated. Lipid ADC was the sole exception as little difference was present between the decanting column and conveyor belt methods. Glencross et al. (2005) also looked at the differences in ADC between the collection column (Cho and Slinger, 1979) and manual stripping (Austreng, 1978) and found a significant difference in ADC levels between the two techniques. Glencross et al. (2005) concluded that manual stripping gave conservative ADC values compared to the decanting column, mainly because of the amount of water exposure faeces experienced for the collection technique. However Glencross et al. (2005) noted that the differences in ADC values were more pronounced for certain ingredients, mainly carbohydrates. Vandenberg and De La Noue (2001) then postulated that endogenous fish products such as blood, urine, and semen could potentially contaminate the samples when manual stripping is conducted. Cho et al. (1985) showed that faeces are collected with incompletely digested materials are mixed with various bodily fluids, the intestinal epithelium, and excess enzymes, which can lead to lower apparent digestibility values. Furthermore, any un-defecated digesta would not be included in any manual stripping ADC values and could explain why there is such a large discrepancy between the three methods (Vandenberg and De La Noue, 2001). Sire and Vernier (1992) showed that proteins taken up in the posterior portion of a fishes intestine could explain the discrepancy between manual stripping and other collection techniques. Therefore, it is possible that when digesta are obtained via manual stripping they are not representative of the entire digestive process.  14  Handling of fish when performing manual stripping can induce a stress response, which has been hypothesized to affect ADC values (Hajen et al., 1993; Belal, 2005). High levels of stress can influence a variety of metabolic factors in fishes including inducing hyperglycemia and higher levels of lactate production, serum cholesterol, skin mucous, and oxygen consumption (Love, 1980). Anesthetics are typically used to minimize handling stress when stripping faeces from fish, but can potentially induce spontaneous defecation, which can dramatically impact ADC values (Spyridakis et al., 1989). This is quite important to note because in certain cases an anesthetic must be used to obtain fish digesta for manual stripping.  2.3.2 Indirect method comparisons Windell et al. (1978) removed digesta from the posterior-most 2.5 cm of the intestine and then submerged it in water for 1, 4, 8, and 16 hour intervals. Most leaching of nutrients occurred during the first hour in the water, while immersion in water for a subsequent hour increased diet digestibility estimates for dry matter, proteins, and lipids by 11.5%, 10.0%, and 3.7%. Percival et al. (2001) demonstrated that leaching was the greatest source of error and leads to overestimates of apparent digestibility values. Therefore it is critical that faeces are removed from the water column as rapidly as possible for any collection method. In contrast to the findings of Windell et al. (1978), Hajen et al. (1993) showed that the majority of nutrient losses for fish faeces in saltwater occur between 6-18 hours of exposure. Similar results were seen in freshwater for proteins and dry matter between 6 – 24 hours (Kabir et al., 1998). It seems reasonable to conclude that faeces obtained from any water collection technique are likely to experience a small degree of nutrient leaching, no matter how quickly they are removed from the water.  15  2.4 Conclusion It is clear that no technique is without problems when it comes to collecting fish faeces for apparent digestibility trials. The two major techniques used to collect fish faeces, manual stripping (direct) and collection of faeces from the water column (indirect), both have strengths and weaknesses. Manual stripping is an excellent technique to use if faeces and feeds do not need to be quantified, and can yield strong results when external markers, such as chromic oxide, are used in conjunction with the stripping (Austreng, 1978). However manual stripping cannot be performed on all species of fish or crustaceans, yields lower ADC values than the column methods, and can be stressful to the fish (Vandenberg and De La Noue, 2001). The major water column collection techniques (Cho and Slinger, 1979; Choubert et al., 1982; Satoh et al., 1992; Hajen et al., 1993) are excellent at reducing levels of distress in fish and can boast high levels of faecal recovery (Choubert et al., 1982). Additionally, and most importantly, faeces can be obtained quite easily from any of these systems. However because the fish faeces may be in the water column for some time, some degree of nutrient loss is likely. If faeces are collected in a timely fashion (Hajen et al., 1993) it is possible to dramatically reduce leaching. Furthermore these techniques may overestimate certain ADC values (Windell et al., 1978; Hajen et al., 1993). Also it may be difficult to construct many of these collection systems in small environments, especially the system developed by Choubert et al. (1982). For large trials involving many fish, using a water column collection method such as those described by Cho and Slinger (1979) or Hajen et al. (1993), is the most practical method to collect fish faeces. Obtaining digesta by dissection should only be performed at the conclusion of trials as fish must be sacrificed. If not limited by time, funding, and space, using the  16  conveyor belt method developed by Choubert and colleagues (1982) would be the best way to determine diet digestibility. When ration levels and feeding frequency rates are monitored, the “Guelph System” (Cho and Slinger, 1979; Hajen et al., 1993) is appropriate for sablefish (Walsh, 1991). In general, the most applicable and plastic techniques to obtain fish faeces are the water column collection methods described by Cho and Slinger (1979), Choubert et al (1982), Satoh et al. (1992), and Hajen et al. (1993).  17  CHAPTER 3: FEEDING FREQUENCY AND WATER TEMPERATURE IMPACT APPARENT DIGESTIBILITY COEFFICIENTS OF SABLEFISH (Anoplopoma fimbria)  3.1 Introduction Aquaculture is the fastest expanding food sector in the world, growing by 7% annually (FAO, 2012). Over-exploited fisheries, increasing consumer demand (FAO, 2012), and technological advancements will help fuel the sector for many years to come. Open-water aquaculture however has been the focus of much criticism around the world in recent years. Concerns regarding open-water aquaculture in British Columbia, Canada, range from waste production, disease control, the effect of escapees on wild fishery populations, and sustainability issues (Ayer and Tyedmers, 2009). While the basis of many of these concerns is beyond the scope of this paper, it is prudent to investigate methods to improve open-water aquaculture in order to address these concerns. Open water integrated multi-trophic aquaculture (IMTA) is a form of polyculture, which focuses on rearing native species inhabiting different trophic levels in close proximity to one another, wherein excess nutrients produced by one species can be utilized by others (Soto, 2009). IMTA systems typically grow finfish (Oncorhynchus, Salmo, Anoplopoma), with shellfish (Mytilus, Haliotis, Crassostrea), algae (Laminaria, Macrocystis), and even echinoderms (Cucumaria) (Troell et al., 2009). A diet is fed to a group of finfish (salmon, sablefish) and the excreted faeces (or soluble nutrients) are in turn utilized by the other co-cultured species (algae, shellfish) within the system (Reid et al., 2011). Ideally IMTA farms attempt to balance waste production with nutrient extraction thereby allowing successful operations to be highly sustainable (Neori et al., 2007), yet still be economically viable (Neori et al., 2004).  18  For IMTA to be successful, information regarding quantity and quality of nutrient transfer between trophic levels is necessary. Knowledge of the dietary and faecal composition for finfish is critical to IMTA because fish, which represent the top trophic level, are the principle drivers, both biologically and economically, for the operation (Soto, 2009). The complexity and scope of some IMTA operations, along with numerous potential species combinations available in any given system, and the amount of baseline research needed in order to create an ecosystem model is staggering. In addition there is a dearth of scientific information on IMTA systems with the majority to date conducted on Atlantic species of fish, shellfish, and algae, with little research conducted on Pacific coast species, such as sablefish (Anoplopoma fimbria). Sablefish, also known as black cod, are deep sea fish that reside along the western continental shelf of North America (Hart, 1973). Sablefish are oily and white-fleshed and can grow to 110 cm in length, 14 kg in weight, and live up to 94 years of age (Kimura et al., 1998). Sablefish are an intriguing species for culture due to their high juvenile growth rates (up to 2.3 mm/day; Shenker and Olla, 1986; Boehlert and Yoklavich, 1985) and profitability (fetching up to $12.35 per kg from 1996-2000; Gislason et al., 2001). Sablefish are currently considered for use in IMTA systems, however more information regarding faecal nutritional content and diet digestibility is required (Reid et al., 2009) before widespread adoption can take place. To calculate the diet digestibility and faecal nutritional content of sablefish, faeces must be collected. Collection of faeces excreted from any aquatic species is problematic because of the leaching of nutrients into the water (Windell et al., 1978; Satoh et al., 1992; Hajen et al., 1993) and because faeces deposited in water can rapidly fragment (Austreng, 1978). Apparent diet digestibility can be determined by collecting faeces using a modified Guelph System (Cho  19  and Slinger, 1979) from fish fed a diet containing an inert marker such as chromium oxide (Cr2O3). This marker is added at a specific ratio in the diet, and measured in the faeces collected from fish fed this diet. The ratio of the marker in faeces, relative to the ratio in the diet, is used to calculate apparent diet digestibility (Maynard and Loosli, 1969). The objective of this research project was to determine the impact of feeding frequency and water temperature on sablefish apparent diet digestibility. Fish feed is the greatest expense for aquaculture farms, and feeding frequency may be manipulated to improve feed efficiencies. Variations in rearing temperatures can also affect diet digestibility and feeding efficiencies (Schurmann and Steffensen, 1997). It was therefore important to understand how feeding frequency and water temperature influence diet digestibility and hence nutrient quality of sablefish faeces. This is critical to assess potential species interaction in an IMTA system. The Canadian Integrated Multi-Trophic Aquaculture Network (CIMTAN) is assimilating knowledge from multiple-disciplines and fields pertaining to IMTA to develop an ecosystem model which can be used for constructing IMTA systems (Chopin, 2011).  3.2 Methods 3.2.01 Experimental fish Juvenile sablefish (~5g) were obtained from Sablefish Canada, Salt Spring Island, BC, and acclimated for two months in indoor 1,000 L tanks at the Department of Fisheries and Oceans, Centre for Aquaculture and Environmental Research, West Vancouver, Canada. Fish were fed a commercial sablefish diet, supplied by Taplow Feeds, North Vancouver, Canada, during this acclimation period. Tanks were supplied with running (6-8 L min-1), sand filtered, oxygenated ambient temperature seawater. For each experiment, fish were randomly distributed 20  into covered 250 L oval, fiberglass tanks supplied with air and provided with a constant photoperiod. Fish were also allowed to acclimate to tank conditions for one week prior to the start of each treatment.  3.2.02 Faecal collection Twelve 250 L tanks were modified to allow for faeces collection, using a modified Guelph collection system (Figure 4) (Cho and Slinger, 1979). Effluent water from the tanks flowed into the collection cylinder through a 3.81 cm diameter PVC pipe. A collection cylinder (1 m in length and 7.62 cm in diameter) with a sloping bottom was attached to each tank. A small opening (diameter of 1.27 cm) was drilled and tapped at the base of the cylinder. A threaded nipple was then screwed into the opening and a short section (20.32 cm) of tubing was inserted on the nipple and clamped. The hydrodynamic properties of this system allowed faeces to accumulate in the collection cylinder and settle at the base of the column, where they could then be collected every morning. After feeding was completed for the day, each tank was thoroughly siphoned to remove uneaten feed. The collection cylinders were cleaned and drained to remove extraneous debris. The following morning, faeces were collected before the fish were fed. Faecal material was collected into Nalgene ® bottles (250 mL) that were attached to each drainage tube at the base of the collection cylinder and filled with a mixture of faeces and saltwater. All bottles were centrifuged for 10 min at 0 ˚C at 5,000 x g. The supernatant was discarded and the resulting faecal pellet frozen at -80 ˚C freezer for six hours, weighed, and stored at -80 ˚C for later analyses.  21  3.2.03 Feed manufacturing All feed was made at the CAER facility to ensure consistency between trials. Mash was obtained from a commercial sablefish feed source (Taplow Feeds, North Vancouver, Canada) and combined with chromic oxide at a ratio of 0.5% Cr2O3/kg feed. Chromic oxide is the predominant inert marker used in terrestrial and fish digestibility studies, and does not move faster than ingesta through the gastrointestinal tract when used at concentrations less than 1% (Tacon and Rodrigues, 1984). Chromic oxide was added slowly to the mash using a large mixer, and mixed for an additional 1 h to ensure consistency throughout the feed. The feed was then steam pelleted with a 3 mm screen and allowed to cool for 3 h. Feed was then top-coated with herring oil in a cement mixer, and allowed to mix for 30 min. Feed was sieved to remove fine particles prior to feedings.  3.2.04 Feeding frequency trial 288 juvenile sablefish (35 ± 5 g) were randomly distributed into each of 12 tanks modified for faeces collection, as described previously. All fish were acclimated to the experimental tanks and the chromic oxide enriched feed for one week prior to the start of the experiment. Four tanks were fed three times daily (9:30AM, 12:00PM, 2:30PM), four were fed twice daily (9:30AM and 2:30PM), and four were fed once daily (12:00PM). The three treatments were randomly assigned to one of three experimental blocks of tanks. All groups were fed the same daily ration (3 % body weight/day) over the duration of the trial. Flow rates for all tanks were set at approximately 6-8 L/min. Faeces were collected in the morning (9:00 AM) before the first feeding using methods described above for 2 weeks.  22  3.2.05 Water temperature trial 264 sablefish (133 ± 17.0 g) were randomly distributed into each of 12 tanks modified for faeces collection as described previously. Two water temperature regimes were compared in this trial. Low temperatures were achieved by chilling incoming seawater in a header tank using a commercial chiller. Eight tanks were supplied with chilled seawater (8 ˚C). 200 watt immersion heaters were used (2 per tank) to heat ambient seawater to 11.5 ˚C in 4 tanks. Flow rates for all tanks were set at approximately 6-8 L/min. The warmer water temperature treatment was separated from the cooler water temperature treatment because the immersion heaters lacked the power to adequately heat tanks to 11.5 ºC when randomly distributed. Temperature, salinity, and dissolved oxygen were monitored twice daily for all tanks. Fish were acclimated to the new water temperatures and the chromic oxide enriched feed for one week prior to the start of the experiment. All fish were fed the same ration (3 % body weight/day) twice daily, first at 9:30AM and then at 2:30 PM. Faeces were collected in the morning (9:00 AM) before the first feeding using the methods as explained above.  3.2.06 Analyses At the time of analyses all faeces were removed from the -80 ˚C freezer, freeze dried over-night, and ground up with a mortar and pestle for subsequent analyses.  3.2.07 Moisture, ash, chromic oxide Freeze-dried faeces (1.5 g/tank) were used to obtain percent moisture, ash, and chromic oxide. In addition, 2.5 g of diet was used to calculate the respective dietary levels in the feed for these same parameters. Samples were weighed in individual crucibles and placed overnight at  23  100 ˚C. Samples were allowed to cool in a dessicator and weighed to obtain moisture values. Afterwards, all samples were placed in a 600 ˚C oven for three hours, cooled and re-weighed to obtain ash values. A 15 mL digestion mixture (150 mL distilled water, 150 mL concentrated H2SO4, 200 mL HClO4, and 10 g MoNa2O4·2H2O) was added to each crucible, and all samples were placed on a 300 ˚C hot plate. Once the samples turned orange they were removed from heat and allowed to cool. The cooled solution was then transferred to 200 mL volumetric flasks, using distilled water to make up the volume, and shaken well. The next day all samples were placed in a spectrophotometer to obtain absorption (at 440 nm) (Fenton and Fenton, 1979). The chromic oxide in the faeces and diet was obtained using the following calculation:  K = 13.6451.  3.2.08 Protein Crude protein content was obtained by using 0.2 g of freeze-dried faeces samples per tank and 0.1 g of diet. All samples were placed in test tubes and combined with 2-3 boiling chips, 2 ground up Pro-Pac (3.5g K2SO4, 0.4g CuSO4) tablets (Alfie Packerrs Inc., 8901 J Street, Suite 10, Omaha, NE, 68127, USA), 15 mL concentrated H2SO4 and allowed to set overnight. The following day all samples were shaken to break up any remaining particulates and then placed in a FOSS 5000 (FIAStar analyzer # 5027). Concentration levels supplied by the machine were given in mg/L. The following formula was used to calculate crude protein content for each sample:  24  K = 6.25  3.2.09 Energy The gross energy of freeze-dried samples of faeces samples (0.25 g/tank) and feed (0.4 g) was obtained by bomb calorimetry (IKA-Werke 5000, cooler # 5001, control # C5003). All samples were placed one at a time in a bomb calorimeter to obtain calorie/gram and Joule/gram readings.  3.2.10 Organic Matter Organic matter dry weight base (DWB) was calculated as follows:  3.2.11 Apparent digestibility coefficient (ADC) calculations The following calculations were used to obtain apparent digestibility coefficients (ADC) for crude protein, gross energy, and organic matter. Protein, energy, organic matter, and chromic oxide were expressed on a dry weight basis (DWB).  25  3.2.12 Statistics ADC values for the feeding frequency trial were examined with one-way ANOVA using the Shapiro-Wilk normality test and Holm-Sidak method. ADC values for the water temperature trial were examined with t-tests assuming equal variance, with the exception of crude protein. Results for the ADCs of crude protein were found to be unequally distributed (F-tests), therefore a Mann-Whitney rank sum test was performed. All values were transferred by performing the arcsin squareroot of the ADCs to determine if data expressed any unusual trends, however none were observed. The computer program (SigmaPlotTM Version 12, Systat Software, Inc., 1735 Technology Drive, Suite 430, San Jose, CA, 95110, USA) was used to analyze data in both trials.  3.3 Results The ADC for crude protein (CP), gross energy (GE), and organic matter (OM) decreased as feeding frequency increased (Table 1). There was a significant difference in all ADCs; CP (One-way ANOVA, p = 0.003), GE (One-way ANOVA, p = 0.043), and OM (One-way ANOVA, p = 0.017). Additionally, all ADC values were grouped together very tightly, often showing no more than a one or two percent difference between all values. All ADC for the water temperature investigation decreased with water temperature (Table 2). There was a significant difference for ADCs between all groups for CP (MannWhitney rank sum test, p = 0.004), GE (t-test assuming equal variance, p = 0.029), and OM (ttest assuming equal variance, p = 0.001). Similar to the values of the frequency fed investigation, all ADC calculations were grouped tightly together for the water temperature trial.  26  3.4 Discussion All ADCs in the feeding frequency trial were highest in the group fed only once per day when compared to the groups fed twice or three times daily. Similar digestibility trends have been seen in rainbow trout (Oncorhynchus mykiss), common carp (Cyprinus carpio), tiger puffer (Takifugu rubripes), and red sea bream (Pagrus major) (Yamamoto et al., 2007; Takii et al., 1997). A potential explanation for the decrease in ADC with higher feeding frequency in sablefish could be attributed to gastrointestinal evacuation rates, the length of the intestine and pyloric caeca, stomach capacity, and fish feeding behaviour. The rate of gastrointestinal evacuation in a major determinant of feed intake (Tyler, 1970; Talbot, 1985; Bromley, 1987) and factors which influence gastrointestinal evacuation include water temperature, particle size, meal size, feed composition, fish size, and previous nutritional history (Fange and Grove, 1979; Smith, 1989; Bromley, 1994). Sablefish are opportunistic, carnivorous fish and may have a slow gastric evacuation rate. Therefore, sablefish may be well adapted to consuming larger, infrequent meals. Sablefish also have a short intestine relative to body length, compared with herbivorous fish species such as tilapia or carp (personal communication with I. Forster), and the length of the intestine and pyloric caeca may play a role in digestibility. The physiological role that the pyloric caeca have is not fully understood in fishes, and it is possible that the caeca may play a small role in the digestion and absorption of nutrients (Ulla and Gjedrem, 1985). It has been shown that a sizeable amount of protein and fat digestion takes place in the anterior half of the small intestine, including the pyloric caeca (Austreng, 1978), particularly in rainbow trout (Dabrowski and Dabrowska, 1981). The size and number of caeca vary between fishes (Bergot et al., 1975), and fishes with many caeca show better feed  27  conversion rates compared to those with lower numbers (Bergot et al., 1981). Ulla and Gjedrem (1985) found that intestine and caeca length do have a significant effect on protein digestibility, while the number of caeca did not have an effect on digestibility. More research on sablefish gastric evacuation rates, intestine and pyloric caeca length, and number of pyloric caeca is needed in order to examine their respective effects on diet digestibility. Altering feeding frequency rates is known to have substantial metabolic effects on fish (Cohn and Joseph, 1959; Fabry, 1967). Grayton and Beamish (1977) showed that energy metabolism in fish is influenced by fish size, water temperature, and diet quality and composition. Infrequent feeding in rats is known as “adaptive hyperliopgenesis,” which leads to increased fat synthesis. This is due to higher activity of adipose tissue enzymes involved with fat synthesis (Mayer et al., 1955; Tepperman and Tepperman, 1958; Cohn and Joseph, 1960; Reeves and Arnrich, 1974). The optimal feeding frequency for salmonids is 1-3 times daily, but may vary due to stomach capacity, digestive rate, and water temperature (Thomassen and Fjaera, 1996). Storebakken and Austreng (1987) showed that increasing daily ration from 50% to 100% satiation increased growth, however any further increases in ration did not result in any additional growth in Atlantic salmon fry. The proportion of lipid attributed to growth increased with the number of feedings in rainbow trout, however the protein content was not affected (Ruohonen et al., 1998). For two species of catfish, when feeding frequency was increased from 2-4 times daily there was a decline in protein utilization (Usmani et al., 2003). For seabass (Dicentrararchus labrax) fry, higher feeding frequencies induced faster growth than continuous feeding of the same amount of feed (Langar, 1997). As previously stated, sablefish are  28  opportunistic fish which may have a large stomach and short intestine (relative to other teleosts), therefore sablefish may respond better to less frequent feedings. Lower feeding frequencies do not necessarily correspond to higher levels of digestibility in all species of fish. Sultana and colleagues (2001) showed that significantly higher growth responses were shown in a treatment group fed four times daily (treatment groups were either fed 2,3,4,5, or 6 times daily). The total food conversion ratio was highest in the group fed twice daily and lowest in the four times daily group (Sultana et al., 2001). However the protein efficiency ratio was highest in the four times daily group and lowest in the twice daily group (Sultana et al., 2001). Additionally the specific growth rate and apparent protein digestibility were highest in the four times daily group (Sultana et al., 2001). Fish reared at warmer water temperatures displayed higher apparent digestibility for crude protein, gross energy, and organic matter. The poikilothermic nature of sablefish may explain these results in part. Warmer water temperatures are known to increase metabolic rates when compared to cooler temperatures (Schurmann and Steffensen, 1997). Similar results have been obtained in Atlantic cod (Gadus morhua) (Schurmann and Steffensen, 1997) and rainbow trout (Azvedo et al., 1998). In general, fish show higher metabolic rates with increased environmental temperatures and juveniles have higher metabolic rates and growth when compared to adults (Schurmann and Steffensen, 1997; Azvedo et al., 1998). Sablefish show extreme skewing towards growth at the expense of other factors when young, especially when compared to similar teleost species (Shenker and Olla, 1986). Collectively these lines of evidence suggest that juvenile sablefish favour growth at the expense of many other metabolic processes.  29  Water temperature has a significant effect on both gut transit time and nutrient digestibility because it has a direct effect on feed intake rates and enzymatic activity (Hidalgo et al., 1999; Temming and Herrmann, 2001; Kofuji et al., 2005). In roach (Rutilus rutilus), increases in water temperature result in heightened enzyme active and feed intake rates (Hardewig and van Djik, 2003). Cooler water temperatures may reduce digestion rates and increase gut transit time (lowering gastrointestinal evacuation rates), thus nutrient digestibility will suffer (Miegel et al., 2010). Protease enzymes in the gut are not as active at lower temperatures, causing lower ADCs for protein (Kofuji et al., 2005). Trypsin levels are lower during the winter months compared to spring months in Atlantic salmon (Einarsson et al., 2005). Furthermore, Alexander and colleagues (2011) showed that higher water temperatures resulted in higher amylase, protease, and hexokinase activities in rohi (Labeo rohita) fingerlings. However McLeese and Stevens (1982) showed that temperature did not have a significant effect on trypsin and chymotrypsin activity in rainbow trout. The growth of salmonids tends to be optimal at 12-17 ºC (Brett, 1971; Koskela et al., 1997a), however they maintain feeding and some growth at temperatures approaching 0C (Kosekla et al., 1997b). Salmonid digestive processes are influenced by temperature (Bendiksen et al., 2003). Salmonids tend to also show increased macronutrient digestibility with increases in temperature (Atherton and Aitken, 1970; Bendiksen et al., 2003). However, sablefish reside in cooler waters and may respond better to cooler temperatures compared to salmonids. More research for sablefish is required in this area. The implications of this research for integrated multi-trophic aquaculture (IMTA) operations are two-fold. Firstly, IMTA operations are open-water systems that undergo water temperature fluctuations throughout the year. It is therefore important to understand the effect of  30  water temperature on finfish faeces apparent digestibility. This research shows that there are differences in ADC for sablefish when reared between 11.5 ºC and 8.0 ºC. Secondly, this research demonstrates that feeding frequency can be reduced without compromising dietary digestibility. This finding may have practical management applications in terms of labour costs associated with IMTA farms. The results from both studies suggest that if high levels of sablefish apparent digestibility are desired, fish should be fed once daily and reared at a warm water temperature. However, the amount of nutrients present in the sablefish faeces for the extractive organics to take up will be less than high feeding frequency rates at cooler water temperatures. For example, sablefish fed once a day and reared at a 11.5 ºC digested 95.1% and 93.6% of the crude protein, respectively, in the feed. Taplow contains 44% crude protein, which means that after digestion, only 2.2% and 2.8% crude protein is present in the faeces. Higher feeding frequencies and cooler water temperatures result in 2.4% and 3.3% crude protein, respectively, in the faeces. In conclusion, these results suggest that for an IMTA operator to balance diet digestibility and maximize the amount of nutrients in the water for uptake by shellfish and algae, optimal feeding frequency rates must be observed in addition to water temperature. I would recommend a heavy feeding regime which results in the most growth for juvenile sablefish, even at the expense of diet digestibility. More research is needed in this regard, however it is likely that an increase in ration, corresponding with an increase in feeding frequency, will result in lower diet digestibility levels. However even if the sablefish are not optimally digesting their feed, more nutrients will reside within the faeces, which can then be taken up by the organic extractive species in the IMTA system. Although the fish are not  31  digesting their feed as well as they could, the nutrients remaining in the faeces are being taken up by the other species in the IMTA system.  32  CHAPTER 4: FIGURES AND TABLES  Figure 1. The locations where pressure is applied to the fish when conducting manual stripping, as described by Austreng (1978) is shown above.  33  Figure 2. The system developed by Choubert et al. (1982). Effluent water, containing faeces (9), flows over a series of moving screens (1). As the unit progresses, faeces dry and are then deposited into a collecting tray (11).  34  Figure 3. The system developed by Hajen et al. (1993). Effluent water flows into the adjacent settling column and faeces collect at the base of the unit.  35  Figure 4. Schematic of faecal collection system when attached to tank. SW = seawater.  36  Table 1. The effect of feeding frequency and rearing temperature on juvenile sablefish (feeding frequency = 35 ± 5g; water temperature = 133 ± 17 g) apparent digestibility coefficients of a commercial diet. ADCs are given as percentages. Low (1 feeding/day, n=4), medium (2 feedings/day, n=4), and high (3 feedings/day, n=4) feeding regimes were used for the feeding frequency study. High (11.5 ºC, n=4) and low (8.0 ºC, n=8) were used for the water temperature study. Mean standard error (MSE) was compiled for the feeding frequency study, and standard error was calculated for the water temperature study. One-way ANOVA were performed for the feeding frequency study (Shapiro-Wilk normality test, Holm-Sidak method). T-tests assuming equal variance were conducted for the water temperature study, except for crude protein. A Mann-Whitney rank sum test was performed for crude protein because data was observed to be unequally distributed (ftest). Significant differences = different letters between treatments, for a particular measurement. Crude Protein  Gross Energy  Organic Matter  Low (n = 4)  95.1 (a)  87.2 (a)  84.0 (a)  Medium (n = 4)  95.1 (a)  86.6 (ab)  83.2 (ab)  High (n = 4)  94.6 (b)  86.1 (b)  82.8 (b)  MSE  0.078  0.269  0.249  p = 0.003  p = 0.043  p = 0.017  High (n = 4)  93.6 (a) 0.326  86.9 (a) 0.427  83.3 (a) 0.449  Low (n = 8)  92.6 (b) 0.071  85.7 (b) 0.239  81.4 (b) 0.197  p = 0.004  p = 0.029  p = 0.001  Trial group A. Feeding Frequency Study  B. Water Temperature Study  37  CHAPTER 5: GENERAL CONCLUSION Sablefish express higher levels of apparent digestibility when fed less frequently and when reared in warmer water temperatures. The results presented in this thesis also agree with the trends shown by many other researchers (Yamamoto et al., 2007; Takii et al., 1997; Schurmann and Steffensen, 1997; Azvedo et al., 1998). These data will be an integral component of an IMTA ecosystem model, which will aid with the proper development of IMTA operations in Canada. Space was a major limitation for both treatments. The laboratory where both experiments took place was quite cramped and the tanks were close to one another. I was unable to modify each tank as well as I would have liked because I did not have enough space, either between, in front of, or above the tanks to fully modify them for faeces collection. Also I was limited by the design and size of the tanks themselves. The tanks used for both studies had a flat bottom, and it would have been ideal to use tanks with sloping bottoms for apparent digestibility experiments. This also meant that I could not use the same tanks for apparent digestibility experiments involving large (1 kilogram +) fish. Furthermore, the amount of feed which I was able to obtain for both studies also limited the duration of each trial and the amount of total (wet) faeces I could collect. Because of this, I was unable to perform any analyses in duplicate (or triplicate), which would have been ideal for such studies. Finally, I was also limited to a small difference in water temperature between treatments for the water temperature investigation due to technical difficulties.  The heater and one (of two) saltwater chillers malfunctioned prior to the water  temperature experiment. I attempted to get both fixed but it would have been impractical from a time and cost perspective. I therefore ordered 8, 200 watt immersion heaters and placed two in each of the four heated tanks. As these heaters were not as effective as the commercial heater, I could not obtain a wide temperature difference between the two treatment groups. 38  Although the water temperatures chosen in the present work were in part a consequence of the technical difficulties and unreliable salt water chillers available, I recommend subsequent work to investigate larger temperature differences between groups (i.e. 5.0ºC, 12.5ºC, and 20.0ºC). Having a larger difference between cooler and warmer temperatures should reinforce the results shown in the water temperature investigation conducted for this paper. Furthermore, a larger temperature range should help disentangle any differences in faecal ADC between cool and warmer waters. I would also recommend a size investigation for sablefish faeces ADC. Sablefish show dramatic feeding changes upon reaching 700-800 g which may affect their digestive capacity. Comparing various sizes of sablefish (i.e. 300g vs. 3000g) could show if there is indeed a difference for apparent digestibility as attributed to size. Preliminary data (S. Pace, unpublished data, 2012) suggests that there is a difference for ADC between small (~430g) and large (~2200g) sablefish for gross energy and organic matter. Juvenile sablefish typically live in warmer, surface water which has an abundance of prey (Sogard and Olla, 1998) and can grow up to 2mm per day in laboratory settings (Shenker and Olla, 1986). Adults are better suited for living in colder temperatures with limited food, yet display a high resistance to starvation (Sullivan and Smith, 1982). Furthermore, sablefish stop growing at approximately ten years of age (McFarlane and Beamish, 1983). Any of these physiological and behavioural differences could have a dramatic impact on faecal ADC, and would be of use for any IMTA operation using different sizes of sablefish. 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